Adiponectin is a potential mediator of synovial fibrosis from early to late knee osteoarthritis

Abstract

Purpose: The endogenous mechanisms associated with synovial fibrosis during osteoarthritis (OA) are not well understood. Fibrosis is characterized by excessive deposition of extracellular matrix, with transforming growth factor beta (TGFB) considered a master regulator. When synovium becomes fibrotic, it is thicker and more rigid, contributing to joint pain and stiffness. Fibrosis in other tissues (e.g. liver) has been associated with various adipokines, which are factors secreted from adipose tissue. Adiponectin is one adipokine previously shown to be dysregulated in OA, and the ADIPOR1 receptor is known to be expressed in synovium, cartilage, and bone. Adiponectin has effects on proteases, cytokines, and inflammatory factors, yet little is known about its role in synovial fibrosis. The aim of this study is to determine the effects of adiponectin in mediating fibrotic changes in the synovium from early to late knee OA. These data will be used to elucidate a potential adiponectin-mediated mechanism for synovial fibrosis in OA progression. Methods: Synovium was obtained from patients immediately following total knee arthroplasty (characterized as late OA by Kellgren-Lawrence grades 3 or 4) or knee arthroscopy (characterized as early OA by Kellgren-Lawrence grades 1 or 2). After 24 hours equilibration in tissue culture, explants were treated with 4 μg/mL adiponectin for 24 hours. For RNA analysis, synovium was snap-frozen in liquid nitrogen, homogenized by Polytron sonication, and subjected to TRIzol® RNA extraction. SYBR Green real-time PCR was used to measure gene expression of common markers of fibrosis, including TGFB, connective tissue growth factor (CTGF), fibronectin (FN1), and collagen type I (COL1A1), and also adiponectin receptor 1 (ADIPOR1). To measure total collagen content and secreted adiponectin levels, tissue culture supernatant was collected and stored at -80C until use. Total collagen was measured using the QuickZyme Biosciences kit and adiponectin was measured using the Abcam SimpleStep ELISA kit. For protein analysis of ADIPOR1, synovial fibroblasts were cultured from synovium and treated with adiponectin for 24 hours then collected in RIPA buffer. Statistical analyses were performed using the Student’s t-test. Results: OA synovium was obtained from 9 patients with late OA and 9 patients with early OA. Among late OA patients, there were 3 females and 6 males between the ages of 55 and 72, all of whom were overweight or obese. Among early OA patients, there were 4 females and 5 males between the ages of 39 and 70, where 5 were overweight or obese and 4 were normal weight. In response to adiponectin treatment, ADIPOR1 showed a trend towards higher expression in late OA compared to early OA synovium, and there was a significant increase in the expression of CTGF and FN1 in late OA, but no significant change to TGFB. Further exploring late OA synovium, there was a positive correlation (r=0.76) between the expression levels of ADIPOR1 and COL1A1 following treatment with adiponectin. Measuring total collagen in the supernatant, levels were 49% higher from late OA synovium as compared to early OA synovium. This corresponded with a 4.3-fold increase in adiponectin in the supernatant from late OA synovium as compared to early OA synovium. Furthermore, protein levels of ADIPOR1 were increased in late OA synovial fibroblasts as compared to early OA synovial fibroblasts, and were further increased in response to adiponectin treatment. Conclusions: Taken together, our results thus far suggest that late OA synovium may produce and secrete higher levels of adiponectin than early OA synovium. This may be activating a fibrotic profile in late OA synovium, including increased collagen production which in part may be mediated through ADIPOR1. Next steps include performing RNA-sequencing of synovium from early and late OA to identify the mechanisms through which adiponectin may be promoting synovial fibrosis.